Abstract LB-368: Exploiting the mechanism of intron-splicing to produce viral vectors harboring toxin genes for cancer therapy

Abstract

Abstract Suicide gene therapy has been widely investigated for the treatment of human immunodeficiency virus (HIV) infection, for controlling graft-versus-host disease, and also for the treatment of cancer. While the production of viral vectors carrying suicide genes such as the herpes simplex virus thymidine kinase (HSV-TK) or cytosine deaminase are easily achievable, the generation of viral vectors containing toxin genes such as diphtheria toxin A fragment (DT-A), Pseudomonas exotoxin (PE), and barnase has always been a challenge very difficult to fulfill due to the extreme toxicity of these toxins. We developed a novel way to produce in insect cells baculoviral and adeno-associated viral (AAV) vectors harboring toxin genes such as DT-A or Barnase by exploiting the different intron-splicing mechanisms between insect and mammalian cells. Since most of the mammalian introns are not functional in insect cells, we cloned two mammalian introns to interrupt the DT-A or Barnase coding sequences and produced recombinant baculoviral and AAV vectors at the normal level as compared with the baculoviral or AAV vectors carrying just the green fluorescent protein (GFP) gene. After transduction of the toxin gene-harboring baculoviral or AAV vectors into mammalian HEK293 cells, the mammalian introns were spliced out and toxins expressed and killed the HEK293 cells. This is the first report so far as we know that the differences in intron-splicing between insect and mammalian cells can be utilized to manufacture viral vectors harboring toxin genes. Our finding indicates that this novel method provides a valuable new tool for the development of toxin-based suicide gene cancer therapy. Bac, baculovirus; AAV, adenoassociated virus; inDTA (hGH), DT-A gene interrupted by human growth hormone intron; inBar (SV40)-GFP, Barnase gene interrupted by SV40 LT antigen intron and fused in-frame with GFP; vg, vector genome copy number; pfu, plaque-forming unit; ND, not done. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-368. doi:1538-7445.AM2012-LB-368